Numerous amplification-based methods for the amplification and detection of target nucleic acids are well known and established in the art. The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of the target sequence (U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 5,804,375). In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from RNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA (U.S. Pat. Nos. 5,322,770; 5,310,652).
PCR reactions generally comprise carrying out multiple cycles of:
(A) hybridizing (annealing) a first primer to a site in a nucleic acid strand at one end of the target nucleic acid sequence, and hybridizing a second primer to a site corresponding to the opposite end of the target sequence in the complementary nucleic acid strand;(B) synthesizing (extending) a nucleic acid sequence from each respective primer; and(C) denaturing the double stranded nucleic acid produced in step (B) so as to form single stranded nucleic acid.Denaturation is generally carried out at from 80 to 100° C., hybridization (annealing) is generally carried out at from 40 to 80° C., and extension is generally carried out at from 50 to 80° C. A typical cycle is denaturation: about 94° C. for about 1 min, hybridization: about 58° C. for about 2 min, and extension: about 72° C. for about 1 min. The exact protocol depends on factors such as the length and sequence of the primers and target sequence, and the enzyme used.
PCR has been adopted for various applications. E.g., GB 2293238 describes methods to reduce non-specific priming and amplifying nucleic acid sequences. Blocking “primers” (or oligonucleotides) are disclosed that produce misalignment and reduce non-specific priming by creating competitive primer annealing reactions to the amplification primers. For example, mixtures of random blocking primers are used that comprise a ddNTP at the 3′ position to prevent initiation of extension reactions. Only correct amplification primers displace their blocking primers and can initiate the amplification reaction.
Methods have been established for using blocking primers that specifically bind to unwanted target oligonucleotide molecules in a sample to prevent amplification thereof in a PCR reaction of unblocked oligonucleotide molecules. Unblocked oligonucleotides can be amplified without further measures to ensure target specificity—in the absence of amplifiable competitive oligonucleotide molecules that are not intended for amplification (US 2002/0076767 A1 and U.S. Pat. No. 6,391,592 B1; WO 99/61661).
A similar method is disclosed in the WO 02/086155, wherein blocking oligonucleotides bound to an undesired template result in premature termination of an elongation reaction. The blocking oligonucleotides bind specifically to one template in a mixture while leaving other templates free for amplification.
U.S. Pat. No. 5,849,497 describes the use of blocking oligonucleotides during a PCR method with a DNA polymerase lacking 5′ exonuclease activity. This DNA polymerase cannot digest the blocking oligonucleotides that prevent amplification. Such a system has been selected to avoid using PNA (peptide nucleic acids) as blocking oligonucleotides. A similar system is described in WO 2009/019008 that however contemplates the use of PNA and LNA, among others, as blocking oligonucleotides.
All these methods have in common that amplification of unwanted templates is specifically suppressed by hybridization of a specific blocking oligonucleotide.
In patent application WO 98/02449 A1 (U.S. Pat. No. 6,090,552) a “triamplification” DNA amplification method is described. It is based on the use of a hairpin primer that is extended and ligated to a blocker. Both primer and blocker bind to one template DNA strand. The second primer binds to the complementary DNA strand. The blocker and one primer are partially complementary with the primer containing a donor and the blocker containing an acceptor moiety for FRET (Fluorescence Resonance Energy Transfer). An extension of the primer and a ligation of an extension product to the blocker leads to a decrease in fluorescence, because they are no longer in close proximity in a blocker primer hybrid. This triamplification method is limited to the use of template DNA and does not relate to RNA methods.
WO 94/17210 A1 relates to a PCT method using multiple primers for both anti-sense and sense strand of a target DNA.
Seyfang et al. [1] describe the use of multiple phosphorylated oligonucleotides in order to introduce mutations into a DNA strand. T4 DNA polymerase, which lacks any detectable strand displacement activity or 5′-3′ exonuclease activity, is used, which is unsuitable for RNA templates.
Hogrefe et al. [2] describe the generation of randomized amino acid libraries with the QuikChange Multi Site-Deirected Mutagenisis Kit. Specific primers containing 3 degenerate nucleotides in the center complementary to a known single stranded target DNA are used. The described kit uses PfuTurbo DNA polymerase which is unsuitable for RNA templates.
The analysis of RNA regularly starts with reverse transcribing RNA into cDNA as DNA is more stable than RNA and many methods exist for analyzing DNA. Whatever protocol is used to analyze the cDNA, it is important that the cDNA generated during reverse transcription (RT) represents the RNA that needs to be analyzed in sequence and concentration as closely as possible.
Reverse transcription is generally carried out using reverse transcriptases. These enzymes require an oligonucleotide primer that hybridizes to the RNA to start (prime) the template dependent polymerization of the cDNA. The two most common priming strategies used are oligo dT priming and random priming.
Oligo dT priming is used for RT of mRNAs that have a poly A tail on their 3′ end. The oligo dT primes the RNA at the 3′ end and the reverse transcriptase copies the mRNA up to its 5′ end. One drawback of this approach is that high quality mRNA is needed as any mRNA degradation will lead to a strong overrepresentation of the 3′ ends of mRNAs.
Even if un-degraded mRNA is used the cDNA molecules may still be truncated due to premature polymerization stop events. A frequent cause is secondary and tertiary structure formation in highly structured RNA regions. Especially when the GC content is high the reverse transcriptase might not read through these regions and thus the cDNA becomes truncated. The likelihood of such events to occur increases the longer the mRNA is that needs to be copied. Therefore oligo dT primed cDNA can show a strong bias towards over-representing the 3′ ends of RNAs. Thus, 3′ end priming suffers from a concentration bias that leads to an increase of sequences at or near the 3′ end with gradually reduced representation of sequences in the direction of the 5′ end (see FIG. 15, triangles, for qPCR measurement of the bias). This is problematic in quantitative approaches, e.g. in the determination of the degree of expression of a particular gene, in difference analysis or in complete expression profiling of a cell.
Approaches have been developed to overcome RNA secondary structure termination especially when long mRNAs need to be reverse transcribed into full length cDNAs. One such method for instance involves a mixture of 2 reverse transcriptases, one highly processive such as MMLV or AMV and mutants thereof, first incubating the reaction mixture at a normal temperature range to allow first strand synthesis plus using a thermostable enzyme composition having reverse transcriptase activity and then incubating the reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand (U.S. Pat. No. 6,406,891). However, buffers for reverse transcriptases contain high concentrations of MgCl2 (3-10 mM) or Mn2+ (e.g for Tth DNA polymerase) and RNA is highly 2unstable and susceptible to breaks and/or degradation at higher temperatures especially in the presence of these divalent cations. The cycling method between two temperatures to bypass secondary structures might also lead to random priming by short RNA fragments that were generated during high temperatures. Such short RNA fragments will be used by MMLV-H or other viral reverse transcriptases as a primer [3]. Again this would lead to a bias in the synthesized cDNA.
Another approach is random priming that has the advantage of hybridizing at multiple locations along the RNA and hence also blocking those sequences from taking part in secondary structure formation. In random priming an oligonucleotide population of random sequence, usually a random hexamer is used to prime the RT anywhere within the template nucleic acid strand. Random priming is used for both, reverse transcription or regular transcription using DNA as template. When product DNA was analyzed it was found that random priming does not result in equal efficiencies of reverse transcription for all targets in the sample [4, 5]. Furthermore there is no linear correlation between the amount of template nucleic acid input and product DNA output when specific targets are measured [4, 5]. Indeed, it has been shown that the use of random primers can lead to overestimate some template copy numbers by up to 19-fold compared to sequence-specific primed templates [6]. Although a lot was speculated about the underlying causes for these phenomena, no conclusive rational has been put forward.